Radiolabelled bisphosphonates and method

ABSTRACT

The present invention relates to  32 P or  33 P-labelled bisphosphonates as radiotherapeutic radiopharmaceuticals. The  32 P- or  33 P-labelled bisphosphonates, which are chemically identical to the unlabelled agent, are expected to target the lesion site in an identical manner, but also deliver a significant radiocytotoxic effect to the surrounding cells. This should result, given the favorable energetics of the β particle emission from the  33 P nuclide, in a loss of proliferative capacity of cells associated with the tumor lesion. The relative stability and in vivo localisation of bisphosphonates makes them good candidates as  32 P/ 33 P delivery vehicles.

This application is a 371 of PCT/EP00/07490 filed Aug. 2, 2000.

FIELD OF THE INVENTION

The present invention relates to ³²P- or ³³P-labelled bisphosphonates asradiotherapeutic radiopharmaceuticals. The ³²P or ³³P-labelledbisphosphonates, which are chemically identical to the unlabelled agent,are expected to target the lesion site in an identical manner, but alsodeliver a significant radiocytotoxic effect to the surrounding cells.This should result, given the favourable energetics of the β particleemission from the ³²P nuclide, in a loss of proliferative capacity ofcells associated with the tumour lesion. The relative stability and invivo localisation of bisphosphonates makes them good candidates as³²P/³³P delivery vehicles.

BACKGROUND OF THE INVENTION

Bisphosphonates are known as palliatives to treat osteosarcoma or bonemetastases associated with carcinoma such as breast or prostate. Theseagents, such as Pamidronate and Clodronate, exert a negative effect uponosteoclasts at the site of the lesion resulting in decreased boneresorption at this site. Bisphosphonates, however, appear to loseactivity with time necessitating repeat administration.

There have been extensive examples of bisphosphonate syntheses in theliterature over the last 25 years. Synthesis of the α-hydroxyl-methylenebisphosphonates, those most commonly studied with respect to bonedisorders, have largely been performed under harsh conditions ofelevated temperatures, often resulting in low-yielding reactions. Thecommon route of synthesis to such compounds involves heating a source ofphosphorus, usually phosphorous acid, with the appropriate carboxylicacid and PCI₃. This synthesis is not ideal for the incorporation of^(32/33)P into a bisphosphonate, due to the safety and radiologicalhazards associated with volatile ^(32/33)PCI₃, and the fact that theprimary role of the PCI₃ is to generate an activated carbonyl compoundrather than as a source of the phosphonate groups. The poor yields oftenobtained from the traditional syntheses are not appropriate forradiolabelling with an expensive radionuclide.

Tris(trimethylsilyl) phosphite, P(OTms)₃, has been isynthesised fromPCI₃ and, alternatively, from phosphorous acid and, subsequently, usedto introduce phosphorus into compounds.

U.S. Pat. No. 3,965,254 describes the use of ^(32/33)P inbisphosphonates for the treatment of bone cancer. It was shown that the³²P or ³³P radionuclide could be targeted to the tumour site. The patentdescribes the incorporation of ³²P into EHDP (disodiumethane-1-hydroxy-1,1-diphosphonate) and its subsequent use in in vivostudies. The synthesis used to generate the radiolabelledbisphosphonates followed the conventional synthesis using ^(32/33)PCI₃.Within the synthetic route the reaction is, at times heated to 145° C.for up to 6 hours and a reflux of 40 hours duration. The final yield wasapproximately 65%. This route of preparation for these compounds is seenas inappropriate for the reasons outlined above.

Accordingly, it is one object of this invention to supply a convenientand improved route of synthesis for radiolabelled bisphosphonates, forthe use in therapeutic treatment of bone metastatic disease.

SUMMARY OF THE INVENTION

In one aspect the invention provides a method of making abisphosphonate, which method comprises reacting a tris(silyl)phosphitewith an activated carbonyl compound and hydrolysing the resultingintermediate according to the reaction scheme:

 P(OX)₃+RCO.Y→Intermediate→RC(OH)(PO.[OH]₂)₂

where

each X is the same or different and is tri-(C₁-C₁₂ hydrocarbyl)silyl,

R is C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, C₂-C₁₂ aryl(including heteroaryl) or substituted variants of these wherefunctionalised groups, if present, are appropriately masked (ie.protected) during the synthesis,

and Y is an activating moiety.

The functional group(s) of the ‘substituted variants’ can be amino(primary, secondary or tertiary), hydroxy, alkoxy or fluorine. R ispreferably C₁-C₁₂ alkyl, C₁-C₁₂ fluoroalkyl or C₁-C₁₂ primary, secondaryor tertiary aminoalkyl or a derivative of these, or a substituted alkylgroup containing nitrogen as part of a heterocyclic ring system. R ismost preferably C₁-C₆ alkyl or C₂-C₉ primary, secondary or tertiaryaminoalkyl. Preferred C₂-C₉ aminoalkyl groups are —(CH₂)_(p)NQ₂ where pis 2 or 3 and Q is H or C₁-C₅ alkyl, with —(CH₂)₂NH₂, —(CH₂)₃NH₂ and(CH₂)₃NMe(pentyl) being especially preferred.

In the starting tris(silyl)phosphite P(OX)₃, X is tri-(C₁-C₁₂hydrocarbyl)silyl, e.g. trialkylsilyl or triarylsilyl, convenientlytrimethylsilyl since derivatised trimethylsilanes are readilycommercially available. Mixed phosphites are possible and may bepreferred.

The starting activated carbonyl compound of formula RCO.Y is preferablyan acid halide, particularly an acid chloride or acid bromide; an acidanhydride; an α-ketophosphonate; or an active ester such as that derivedfrom N-hydroxysuccinimide. Hence the activating moiety Y can be: aleaving group such as halogen (especially Cl or Br), or an acidanhydride linkage (RCO)₂O, or an active ester, examples of which arewell known to those skilled in the art. Alternatively, Y can be aphosphonate —PO(OR′″)₂, ie. RCOY may be an α-ketophosphonate.Preparation of bisphosphonates has also been achieved using an activeester derived from 2-hydroxypyridine and also acid anhydrides. Activeesters derived from pentafluorophenol and hydroxybenztriazole are alsopossible. These reactions show how the increased nucleophilicity of thesilylated phosphites (compared with trialkyl phosphites) permits the useof much less activated carbonyl compounds in the formation of thedesired products.

When Y is a leaving group, the Intermediate generally has the formulaRC(OZ)(PO.[OX]₂)₂ where Z is H or X. When RCOY is an α-ketophosphonate,an addition reaction rather than a substitution reaction may take place,e.g.:

P(OX)₃+RCO.PO(OR′″)₂→RC(OZ)(PO)[OX]₂)(PO[OR′″]₂)→RC(OH)(PO[OH]₂)₂

where R′″ is C₁₋₁₂ alkyl or X (by treatment of the —O-alkyl system withTmsBr or Tmsl).

The R group is chosen to provide a desired substituent on thehydroxymethane-bisphosphonate unit.

Various bisphosphonate drugs R¹CR²(PO[OH]₂)₂ have been commercialised asfollows:

Name R¹ R² Etidronate OH —CH₃ Ibandronate OH —CH₂CH₂NMe(pentyl)Alendronate OH —(CH₂)₃NH₂ Pamidronate OH —(CH₂)₂NH₂ Clodronate Cl Cl

Preferably the tris(silyl)phosphite contains ³²P or ³³P, either at 100%abundance or at least at an artificially high isotopic abundance of e.g.at least 1%. Preferably 2 molar equivalents of the tris(silyl)phosphiteare reacted with 1 molar equivalent of the activated carbonyl compound.Although use of elevated temperatures is possible, the reaction is foundto go in high yield at ambient temperature, thus providing a convenientroute for introducing ³²P or ³³P into a bisphosphonate molecule ofchoice.

Dry aprotic solvents such as diethyl ether or tetrahydrofuran can beused to help facilitate handling of the reactants. Reaction times dependon the leaving group and on the quantity of solvent used. With modestquantities of solvent, reaction times are typically 10 to 15 min with anacid chloride or anhydride but it can require heating for several hourswith less activated carbonyl compounds.

There results an intermediate having the formula RC(OZ)(PO.[OX]₂)₂ orRC(OZ)(PO)[OX]₂)(PO[OR′″]). In general these intermediates are believednovel and form further aspects of this invention. They may readily behydrolysed to the desired bisphosphonates by the addition of excessmethanol or water at elevated or preferably ambient temperature. Some ofthe resulting bisphosphonates are new compounds.

Thus the invention also provides radiolabelled bisphosphonates of theformula:

R″C(OH)(^(n)PO[OH]₂)₂

where

n is at least partly 32 or 33 whereby the compound contains anartificially high proportion of ³²P or ³³P,

and R″ is C₁-C₁₂ primary, secondary or tertiary aminoalkyl or aderivative of these, or a substituted alkyl group containing nitrogen aspart of a heterocyclic ring system. R″ is preferably C₂-C₉ primary,secondary or tertiary aminoalkyl, most preferably —(CH₂)_(p)NQ₂ where pis 2 or 3 and Q is H or C₁-C₅ alkyl, with —(CH₂)₂NH₂, —(CH₂)₃NH₂ and(CH₂)₃NMe(pentyl) being especially preferred.

It may be advantageous to provide one of the starting reagents, e.g. thetris(silyl)phosphite POX₃ or the activated carbonyl compound RCOY, inimmobilised form. This facilitates purification of the startingcompound, which may be particularly useful when an isotopically labelledphosphite is used, and also facilitates purification of the desiredbisphosphonate product.

The radiolabelled bisphosphonates of the present invention have usefulproperties as palliatives to treat osteosarcoma or bone metastasisassociated with carcinoma such as breast or prostate, with theadditional advantage of comprising an artificially high isotopicabundance of ³²P or ³³P for targeting either bone metastasis or tumoursor other diseases so that may be treated with radiotherapy.

Also included within the scope of the invention are radiolabelledphosphorus compounds of the formula

R′_(q) ^(n)P(OX)_(3−q)

where

R′ is C₁-C₁₂ alkyl or C₂-C₁₂ aryl,

q is 0, 1 or 2,

n is at least partly 32 or 33 whereby the compound contains anartificially high proportion of ³²P or ³³P,

each X is the same or different and is tri-(C₁-C₁₂ hydrocarbyl)silyl.

When q is 0, these are radiolabelled trialkylsilylphosphites, useful asstarting materials in the method of the invention described above.

DETAILED DESCRIPTION OF INVENTION EXAMPLE 1 Preparation ofTris(trimethylsilyl) Phosphite (compound 1)

Triethylamine (40.4 g, 40.4 mmol) was added dropwise to a mechanicallystirred solution of phosphorous acid (16.5 g, 201 mmol) andchlorotrimethylsilane (44 g, 405 mmol) in diethyl ether (1200 cm³).Immediately, a white precipitate of triethlyamine hydrochloride formed.After the addition was complete (2 h) the mixture was stirred for afurther 2 hours. The insoluble salt was removed by filtration and thefiltrate concentrated to about 200 cm³. The small quantity of additionalprecipitate formed during this process was subsequently filtered off andthe filtrate evaporated to give a cloudy liquid. The product waspurified by distillation under reduced pressure, the fraction boiling inthe range 45-50° C. at 0.1 mbar being collected. The bis(trimethylsilyl)phosphite was isolated as a colourless oil (35 g, 77%). ³¹P NMR: δ−12.8.Chlorotrimethylsilane (20 g, 185 mmol) was added dropwise to a stirredsolution of bis(trimethylsilyl)phosphite (30 g, 132 mmol) andN,N-diethyltrimethylsilylamine (20 g, 137 mmol) and the mixture stirredat room temperature for 1 h. The solid formed was removed by filtrationand the filtrate distilled in vacuo. A colourless fraction boiling at77-80° C. at 0.1 mbar was collected (30 g, 75%). ³¹P NMR: δ+116.3.

EXAMPLE 2 Synthesis of Hydroxyethylidene Bisphosphonic Acid (compound 2)

Tris(trimethylsilyl) phosphite (2 molar equiv.) in an appropriate drysolvent was added dropwise to a stirred solution of acetyl chloride (1molar equiv.) in an appropriate dry solvent at room temperature and themixture stirred for 10 minutes. Excess methanol was then added and themixture stirred for 5 minutes to decompose the silyl esters. Thevolatile components in the reaction mixture were then removed underreduced pressure to leave the title compound in good purity.

It is anticipated that this reaction can be performed using ³²P(OTms)₃or ³³P(OTms)₃ in order to introduce a ³²P label or a ³³P label.

EXAMPLE 3 Synthesis of 4-Amino-1-hydroxybutane-1,1-bisphosphonic Acid(compound 3)

A solution of benzylchloroformate (9.4 g, 5.5 mmol) in acetone (15 ml)was added dropwise to a mixture of 4-aminobutanoic acid (5.2 g, 5 mmol),sodium bicarbonate (8.6 g, 10 mmol), water (25 ml) and acetone (25 ml)causing vigorous effervescence. The solution was allowed to stirovernight. The acetone was removed using a rotary evaporator to leave anaqueous phase which was washed with ether (2×25 ml). The resultingaqueous layer was acidified to pH2 with dilute hydrochloric acid andthen extracted with dichloromethane (3×50 ml). The dichloromethaneextracts were combined, dried over magnesium sulphate and volatilecomponents removed under reduced pressure to leave4-[(benzyloxycarbonyl)amino]butanoic acid as a white solid in a goodstate of purity. This material was used without further purification.

A solution of dicyclohexylcarbodiimide (2.5 g, 12 mmol) in drytetrahydrofuran (10 ml) was added in one portion to a stirred solutionof 4-[(benzyloxycarbonyl)amino] butanoic acid (2.4 g, 10 mmol) andN-hydroxysuccinimide (1.5 g, 13 mmol) in dry tetrahydrofuran (10 ml).After about 20 minutes dicyclohexylurea began to precipitate from thesolution. Stirring was continued for 16 hours and the resulting solutionwas then filtered. The insoluble white solid was removed and washed withtetrahydrofuran (20 ml) and the washings combined with the filtrate.Volatile components were removed from the tetrahydrofuran solution invacuo to leave a white waxy solid. This residue was purified bychromatography on silica using ethyl acetate as the eluant. Removal ofthe solvent under reduced pressure gave a viscous colourless oil thatwas triturated with dry ether to produce a white powder.

¹H NMR: δH (CDCl₃); 7.35-7.27 (5H, m), 5.09 (3H, br), 3.30 (2H, br q),2.79 (4H, s), 2.66 (2H, t), 1.97 (2H, pentet).

Tris(trimethylsilyl)phosphite (900 mg, 3 mmol) was added to a solutionof succinyl 4-[(benzyloxycarbonyl) amino]butanoate (340 mg, 1 mmol) indioxane (0.5 ml) and the mixture warmed at 65-70° C. with the exclusionof moisture until shown by ³¹P NMR spectroscopy to be completed.Bromotrimethylsilane (0.5 g, 3.25 mmol) was added and the solutionstirred for ca 1 h at room temperature. Volatile components were removedin vacuo to leave a viscous oil. Methanol (20 ml) was added and theresulting mixture stirred for 1 h. The solid product was then filteredoff. More product precipitated on addition of acetone to the filtrateand this was added to the filtered solid. After drying the requiredbisphosphonate (compound 3) was obtained as a white solid (130 mg, 52%).

δ_(P) 19.5, δ_(C) (D₂O) 73.0 (t, J_(PC)=138 Hz), 39.9 (s), 27.0 (t,J_(PC)=7 Hz),

δ_(H)(D₂O) 2.76 (2H, m, CH₂N), 1.75 (4H, m, CH₂CH₂).

EXAMPLE 4 Solid Phase Synthesis of 4-Amino-1-hydroxybutane-1.1bisphosphonic Acid (compound 3)

To 0.5 g of 2-chlorotrityl chloride resin suspended in anhydrousdimethylformamide was added 5 molar equivalents of β-alanine ethyl esterhydrochloride and 10 molar equivalents of triethylamine. The resin wasagitated for 48 hours and then washed exhaustively with dichloromethaneand dimethylformamide. The ester was cleaved by the addition of KOH indioxan and agitation for 8 hours. The resin was washed thoroughly withmethanol and dichloromethane after filtration. Addition of N-hydroxysuccinimide (5 molar equiv.) and dicyclohexylcabodiimide (5 molarequiv.) in dichloromethane with stirring for 24 hours afforded theactivated ester. After filtration the resin was washed with methanol anddichloromethane. To this resin in dichloromethane was addedtris(trimethylsilyl) phosphite (5 molar equiv.) and agitated for 48hours at room temperature. The resin was then washed withdichloromethane and methanol followed by treatment with 10%trifluoroacetic acid in dichloromethane to cleave the product.

Synthetic route to an amino-bisphosphonate on a solid support

What is claimed is:
 1. A method of preparation of a bisphosphonate,which method comprises reacting a tris(silyl)phosphite with an activatedcarbonyl compound and hydrolysing the resulting intermediate accordingto the reaction scheme: P(OX)₃+RCO.Y→Intermediate→RC(OH)(PO.[OH]₂)₂where each X is the same or different and is tri-(C₁-C₁₂hydrocarbyl)silyl, R is C₁-C₁₂ primary, secondary or tertiaryaminoalkyl, and Y is an active ester, N-hydroxysuccinimide or PO(OR′″)₂,where R′″ is C₁₋₁₂ alkyl or X.
 2. The method of claim 1, wherein X ofP(OX)₃ is trimethylsilyl.
 3. The method of claim 1, wherein thetris(silyl)phosphite P(OX)₃ has artificially high proportion of ³²P or³³P.
 4. The method of claim 1, performed at ambient temperature.
 5. Themethod of claim 1, wherein Y is an active ester or N-hydroxysuccinimideand the Intermediate has the formula RC(OZ)(PO.[OX]₂)₂ where Z is H orX.
 6. The method of claim 1, wherein Y is PO(OR′″)₂ and the Intermediatehas the formula RC(OZ)(PO)[OX]₂)(PO[OR′″]₂).
 7. The method of claim 1,wherein the tris(silyl)phosphite P(OX)₃ or the activated carbonylcompound RCO.Y is immobilised on a solid support.
 8. A radiolabelledphosphorus compound of the formula R′_(q) ^(n)P(OX)_(3−q) wherein R′ isC₁-C₁₂ alkyl or aryl, q is 0, 1 or 2, n is at least partly 32 or 33whereby the compound has an artificially high proportion of ³²P or ³³P,X is tri-(C₁-C₁₂ hydrocarbyl)silyl, and may be the same as or differentfrom other X's.
 9. The radiolabelled phosphorus compounds of claim 8,where q is 0 and X is trimethylsilyl.
 10. A radiolabelled biphosphonateof the formula R″C(OH)(^(n)PO[OH]₂)₂ wherein n is at least partly 32 or33 whereby the compound has an artificially high proportion of ³²P or³³P, and R″ is C₁-C₁₂ primary, secondary or tertiary aminoalkyl or aderivative of these, or a substituted alkyl group having nitrogen aspart of a hetreocyclic ring system.
 11. The radiolabelled biphosphonateof claim 10, where R″ is —(CH₂)_(p)NQ₂, p is 2 or 3, and Q is H or C₁₋₅alkyl.
 12. A method of treating a patient suffering from osteosarcoma orbone metastases, which method comprises administering to the patient aneffective amount of the radiolabelled biphosphonate of claim
 11. 13. Acompound of the formula RC(OZ)(PO[OX]₂)₂ or RC(OZ)(PO[OX]₂)(PO[OR′″])wherein X is tri-(C₁-C₁₂ hydrocarbyl)silyl, and may be the same as ordifferent from other X's, Z is H or X, R′″ is C₁-C₁₂ alkyl or X, and Ris C₁-C₁₂ primary, secondary or tertiary aminoalkyl.
 14. The compoundsof claim 13, having an artificially high proportion of ³²P or ³³P.